Causes Of Mismatch Repair Deficiency Research Articles

Comprehensive molecular assessment of mismatch repair deficiency in Lynch associated ovarian cancers using next generation sequencing panel

ObjectivesAbnormalities in mismatch repair have been described in ovarian cancer, but few studies have examined the causes of mismatch repair deficiency (MMRd). To address this, we completed targeted mutational and...

The impact of cause of mismatch repair deficiency and other molecular markers on clinical outcomes with the use of durvalumab in advanced endometrial cancer in the phase 2 PHAEDRA trial (ANZGOG1601).

5604 Background: In the single arm phase 2 PHAEDRA trial, MMR deficiency (dMMR) was predictive of response to durvalumab (1500mg IV Q4W), with an objective tumor response rate (OTR; defined by iRECIST) of 47% in dMMR compared with 3% in MMR-proficient (pMMR) advanced endometrial cancer (AEC). This substudy of the PHAEDRA trial investigates MMR molecular subtypes and other genomic tumor features and their correlation with treatment outcomes. Methods: Testing was performed to determine molecular subtypes of dMMR, including germline pathogenic variants in DNA MMR genes (Lynch syndrome), somatic biallelic MMR gene inactivation through somatic mutation and somatic hypermethylation of the MLH1 gene promoter. DNA from formalin-fixed paraffin-embedded tumor tissue and matched peripheral blood was available from 41/71 (25 dMMR, 16 pMMR) participants for testing on a custom capture-based 298-gene targeted panel (2.005Mb) including the MMR genes and key somatic AEC driver genes. The derived tumor genomic features included microsatellite instability (MSI), tumor mutational burden (TMB), COSMIC v.3.2 tumor mutational signatures and insertion/deletion (Indel) somatic mutation count. Results: Of the 71 patients recruited, 35 were dMMR and 36 were pMMR. Median follow-up was 44 vs 52 months in dMMR vs pMMR participants, respectively. The dMMR molecular subtypes were 4 (11.4%) Lynch syndrome, 4 (11.4%) somatic MMR mutation, 25 (71.4%) MLH1 methylated and 2 (5.7%) dMMR-uncategorised. The OTR rate was 100% (4/4; 95%CI 40-100%) for Lynch, 75% (3/4; 95%CI 22-99%) for somatic MMR mutations and 40% (10/25; 95%CI 22-61%) for MLH1 methylated groups. The median TMB (assessed in 41/71) was higher in those with a confirmed radiological response (37, IQR:26-50) vs non-responders (16, IQR:9-25; p < 0.001). Within the MLH1 methylated group, TMB was also higher in responders vs non-responders (40 v 21; p = 0.03). Somatic mutations in KRAS, PTEN, PIK3CA, ARID1A and TP53 were not associated with OTR rate. Conclusions: dMMR- MLH1 methylated AEC demonstrated greater heterogeneity in OTR to single agent durvalumab than the dMMR-Lynch and dMMR-somatic MMR mutation molecular subtypes. Higher TMB was seen in responders, and specifically within dMMR- MLH1 methylated responders, compared to non-responders. Clinical trial information: ANZGOG1601.

MLH1/PMS2-deficient Endometrial Carcinomas in a Universally Screened Population: MLH1 Hypermethylation and Germline Mutation Status.

MLH1/PMS2 loss due to epigenetic hypermethylation of the MLH1 promoter is the most common cause of mismatch repair deficiency in endometrial carcinoma, and typically provides reassurance against an associated germline mutation. To further characterize the genetic features of MLH1/PMS2-deficient endometrial cancers, the departmental database was searched for cases with dual MLH1/PMS2 loss and retained MSH2/6 expression which underwent MLH1 hypermethylation testing. Genetic testing results were obtained when available. One hundred seventeen endometrial cancers met inclusion criteria: 100 (85%) were MLH1-hypermethylated, 3 (3%) were low-level/borderline, 7 (6%) were nonmethylated, and 7 (6%) were insufficient for testing. Sixteen cases (12 MLH1-hypermethylated, 3 nonmethylated, and 1 insufficient for testing) underwent germline testing, 6 of which (37.5%) demonstrated germline variants of unknown significance (VUS) (MSH6, PMS2, POLD1, BRIP1, RAD51D, CHEK2) but no known deleterious mutations. Notably, however, the patients harboring the MSH6 and PMS2 germline VUS had clinical features concerning for Lynch syndrome. One nonmethylated, germline-normal case underwent somatic tumor testing, and demonstrated a somatic MLH1 mutation. In summary, MLH1-hypermethylation accounts for the vast majority of MLH1/PMS2-deficient cancers in a universally screened population, although MLH1 somatic and germline mutations can occur. Occasionally, patients with MLH1-hypermethlated tumors also bear germline VUS in other mismatch repair genes as well as genes implicated in other hereditary cancer syndromes, but their clinical relevance is unclear. Family and personal cancer histories must always be evaluated to determine the need for germline testing in women with loss of MLH1/PMS2, even in the setting of hypermethylation.

An Integrative DNA Sequencing and Methylation Panel to Assess Mismatch Repair Deficiency

Clinical testing for mismatch repair (MMR) deficiency often entails serial testing of tumor and constitutional DNA using multiple assays. To minimize cost and specimen requirements of MMR testing, we developed an integrated targeted sequencing protocol (termed MultiMMR) that tests for promoter methylation, mutations, copy number alterations, copy neutral loss of heterozygosity, and microsatellite instability from a single aliquot of DNA. Hybrid capture of DNA-sequencing libraries constructed with methylated adapters was performed on 142 samples (60 tumors and 82 constitutional samples) from 82 patients with MMR-associated colorectal, endometrial, and brain cancers as well as a synthetic DNA mix with 11 known mutations. The captured material was split to enable parallel bisulfite and conventional sequence analysis. The panel targeted microsatellite regions and 13 genes associated with MMR, hypermutation, and hereditary colorectal cancer. MultiMMR recapitulated clinical testing results in 23 of 24 cases, was able to explain MMR loss in an additional 29 of 48 patients with incomplete or inconclusive testing, and identified all 11 MMR variants within the synthetic DNA mix. Promoter methylation and microsatellite instability analysis found 95% and 97% concordance with clinical testing, respectively. We report the feasibility for amalgamation of the current stepwise and complex clinical testing workflow into an integrated test for hereditary and somatic causes of MMR deficiency.

Comprehensive molecular assessment of mismatch repair deficiency in Lynch-associated ovarian cancers using next-generation sequencing (NGS) panel.

1523 Background: Abnormalities in mismatch repair (MMR) gene may be the result of pathogenic germline (Lynch syndrome) and somatic mutations as well as epigenetic events. Abnormalities in MMR have been described in non-serous/non-mucinous ovarian cancer (OC) but few studies have examined the causes of these MMR defects (MMRd). To address this, we have completed targeted mutational and methylation sequencing on MMRd OC cases. Methods: Women with newly diagnosed non-serous/mucinous OC (N = 215) were prospectively recruited from three cancer centers in Ontario, Canada between 2015-18. Tumors were reflexively assessed for MMR protein expression by immunohistochemistry. Tumor DNA was extracted from macrodissected MMRd cases and MMR-intact (MMRi) controls following pathology review. Matched tumor-normal samples were run on a custom NGS panel to identify germline and somatic mutations, copy number variants, rearrangements and promoter methylation in MMR and associated genes. Results: Of the 215 women enrolled in our study, 185 (86%) had OC alone and 30 (14%) had synchronous OC and endometrial cancer. Twenty-eight (13%) cases were MMRd, 11 of which were synchronous. The MMRd cohort had median age of 52.5 years, with mostly stage I (N = 14; 50%), grade 1 or 2 disease (N = 18; 64%) with endometrioid histotype (N = 18; 64%). One patient had recurrence after median follow-up of 33.6 months (13.2-93.6). There was no significant difference in overall/progression-free survival between the MMRd and MMRi patients. Using the NGS panel, Lynch syndrome (LS) was detected in 39% of MMRd cases (11/28; 7 OC and 4 synchronous): 7 MSH6, 2 MLH1, 1 PMS2, and 1 MSH2. Clinical germline sequencing was performed on all cases and verified panel findings. An explanation for the observed MMR phenotype was available for 18/20 deficient cases, including 9/10 MLH1−/PMS2− (7 somatic methylation, 1 bi-allelic somatic deletion, 1 germline mutation), 0/1 PMS2−, 6/7 MSH6− (6 germline mutations) and 2/2 MSH2−/MSH6− (1 germline mutation, 1 bi-allelic somatic mutation). Concordance between clinical and research panel sequencing results was 90%. None of the germline mutations were missed by the panel. Conclusions: Use of our custom NGS panel allows for the streamlined assessment of hereditary and somatic causes of MMR deficiency in OC and may be an attractive screening strategy for LS in this population.

Endometrial cancer risk and survival by tumor MMR status.

ObjectiveThe risk of developing endometrial cancer (EC) and/or survival following a diagnosis of EC might differ by tumor DNA mismatch repair (MMR) status. We assessed the association between tumor MMR status (classified as MMR-proficient, somatic MMR-deficient, germline MMR-deficient) and the risk of developing EC and survival following a diagnosis of EC.MethodsWe analyzed data from women who participated in the Australian National Endometrial Cancer Study (ANECS) conducted between 2005 and 2007. Risk analyses (698 cases/691 population controls) utilized sociodemographic and lifestyle information obtained from telephone interviews at recruitment. For survival analyses (728 cases), patients' clinical data was abstracted from medical records, and survival data were obtained via linkage with the Australian National Death Index. We used logistic regression analysis to evaluate the associations between tumor MMR status and EC risk, and proportional hazards models to perform survival analyses with adjustment of known prognostic factors.ResultsEstablished risk factors for EC did not differ significantly by tumor MMR status. In analyses including all EC subtypes, overall and EC-specific survival did not differ by tumor MMR status. Among women with the most common endometrioid subtype, EC-specific survival was worse for women with somatic MMR-deficient EC compared to women with MMR-proficient EC (hazard ratio [HR]=2.18; 95% confidence interval [CI]=1.19–4.01).ConclusionThe risk of EC is not associated with MMR status. Accurate separation of germline from somatic causes of MMR deficiency suggests that patients with endometrioid subtype somatic MMR-deficient tumors have poorer EC-specific survival than those with MMR-proficient tumors, after accounting for other prognostic factors.

EPCAM germline and somatic rearrangements in lynch syndrome: identification of a novel 3′EPCAM deletion

3'EPCAM (Epithelial Cell Adhesion Molecule) genomic rearrangements can be a cause of mismatch repair deficiency in rare Lynch syndrome families. 3'EPCAM deletions include the polyadenylation signal and might result in promoter hypermethylation of the centromeric MSH2 gene in cis. A somatic rearrangement in trans affecting MSH2 is responsible for the final mismatch repair deficiency in the corresponding tumors but the mechanisms are not well documented. In this report two germline 3'EPCAM deletions are described together with the corresponding somatic mutations in the patient's colorectal tumors. Mutation and breakpoint analysis resulted in the identification of one novel (c.556-531_*872del) and one known EPCAM deletion (c.859-689_*14697del). Both deletions resulted from Alu mediated homologous recombination causing aberrant EPCAM-MSH2 fusion transcripts. The colorectal tumors of the deletion carriers were MSI-high. Strong hypermethylation of the MSH2 promoter was measured. Analysis of somatic genomic rearrangements showed a 4 Mb deletion including the EPCAM, MSH2 and MSH6 genes in one tumor and copy neutral loss of heterozygosity in the EPCAM-MSH2 region in the other tumor. This indicates that hemi- and homozygous hypermethylation of the MSH2 promoter and hence complete silencing of MSH2 expression was responsible for the mismatch repair deficiency in both colorectal tumors.

Abstract 2619: Relevance of mismatch repair deficiency in ovarian cancer

Abstract Ovarian cancer is the leading cause of death in gynecologic malignancies in the United States with most patients presenting with late stage disease; patients receive the same treatment regardless of histology or tumor characteristics. The mismatch repair (MMR) molecular pathway is well-defined and known to be involved in up to 20% of ovarian cancers; however, association of deficient v proficient MMR status (dMMR v pMMR status) with risk factors and outcome is not well understood. We are conducting extensive molecular testing of the MMR genes MLH1, MSH2, MSH3, MSH6, and PMS2 on Mayo Clinic invasive epithelial ovarian cancer cases. First, the prevalence of dMMR tumors will be estimated in ovarian cancer cases (n=496) based on loss of expression of MLH1, MSH2, MSH3, MSH6, and PMS2 determined by immunohistochemistry staining of primary antibodies using negative and positive controls. Staining of tissue microarrays (TMAs) is complete; triplicate cores will be scored independently for presence or loss of protein expression by a board-certified pathologist. Second, for those dMMR cases, the possible cause of MMR deficiency will be evaluated considering germline and somatic mutations. The loss of expression of each protein will be characterized as germline if a deleterious mutation is present in peripheral blood or somatic if the pathogenic mutation is only present in tumor tissue. In cases with MLH1 loss somatic hypermethylation will also be considered. Analysis of germline mutation data will include all 496 cases and somatic mutations data will include a subset of cases tested with frozen tumor DNA (n=292) using Fluidigm access array targeted resequencing. Third, the clinical features of dMMR disease will be compared to those of pMMR; in particular, age at diagnosis, histopathology, and disease outcome. Among cases in this analysis, the majority have high-grade serous disease with a mean age at diagnosis of 61 years, about half had recurrence or progression (n=243) of which two-thirds (n=167) are deceased. Finally, known and suspected ovarian cancer risk factors (family history, smoking, body mass index, parity, use of hormone therapy, and genetic variation in susceptibility loci at 2q31, 3q25, 8q24, 9p22, 17q21, and 19p13) will be analyzed by MMR phenotype. Risk factor questionnaire data is available on 462 cases (93%), as well as 737 population controls with germline genome-wide genotype data. About half of the cases (n=231) report a 1st or 2nd degree family member with breast or ovarian cancer. Given the limited knowledge about ovarian cancer etiology and the poor prognosis of advanced disease, it is very important to study risk factors with this molecular subtype. The ovarian cancer dMMR phenotype may have distinct clinicopathological features that could guide risk stratification and be useful in diagnostics and therapeutics. This is the largest study characterizing MMR in an ovarian cancer population to date. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2619. doi:1538-7445.AM2012-2619

MSH6 and MUTYH Deficiency Is a Frequent Event in Early-Onset Colorectal Cancer

Early-onset colorectal cancer (CRC) is suggestive of a hereditary predisposition. Lynch syndrome is the most frequent CRC hereditary cause. The MUTYH gene has also been related to hereditary CRC. A systematic characterization of these two diseases has not been reported previously in this population. We studied a retrospectively collected series of 140 patients ≤50 years old diagnosed with nonpolyposis CRC. Demographic, clinical, and familial features were obtained. Mismatch repair (MMR) deficiency was determined by microsatellite instability (MSI) analysis, and immunostaining for MLH1, MSH2, MSH6, and PMS2 proteins. Germline MMR mutations were evaluated in all MMR-deficient cases. Tumor samples with loss of MLH1 or MSH2 protein expression were analyzed for somatic methylation. Germline MUTYH mutations were evaluated in all cases. BRAF V600E and KRAS somatic mutational status was also determined. Fifteen tumors (11.4%) were MSI, and 20 (14.3%) showed loss of protein expression (7 for MLH1/PMS2, 2 for isolated MLH1, 3 for MSH2/MSH6, 7 for isolated MSH6, and 1 for MSH6/PMS2). We identified 11 (7.8%) germline MMR mutations, 4 in MLH1, 1 in MSH2, and 6 in MSH6. Methylation analysis revealed one case with somatic MLH1 methylation. Biallelic MUTYH mutations were detected in four (2.8%) cases. KRAS and BRAF V600E mutations were present in 39 (27.9%) and 5 (3.6%) cases, respectively. Loss of MSH6 expression is the predominant cause of MMR deficiency in early-onset CRC. Our findings prompt the inclusion of MSH6 and MUTYH screening as part of the genetic counseling of these patients and their relatives.

Mismatch repair deficiency in hematological malignancies with microsatellite instability.

Mutations in human mismatch repair (MMR) genes are the genetic basis for certain types of solid tumors displaying microsatellite instability (MSI). MSI has also been observed in hematological malignancies, but whether these hematological malignancies are associated with MMR deficiency is still unclear. Using both biochemical and genetic approaches, this study analysed MMR proficiency in 11 cell lines derived from patients with hematological malignancies and demonstrated that six out of seven hematological cancer cell lines with MSI were defective in strand-specific MMR. In vitro complementation experiments, using characterized MMR mutant extracts or purified proteins, showed that these hematological cancer cells were defective in either hMutS(alpha) (a heterodimer of hMSH2 and hMSH6) or hMutL(alpha) (a heterodimer of hMLH1 and hPMS2). Furthermore, cell lines deficient in hMutS(alpha) showed large deletions or point mutations in hMSH2, while those deficient in hMutL(alpha) exhibited point mutations in hMLH1 or a lack of expression of hPMS2. From these results, we conclude that, as in solid tumors, hematological malignancies with MSI are also associated with MMR deficiency, and that the cause of MMR deficiency in these cell lines is due to a defective MutS(alpha) or MutL(alpha). We also report here, for the first time, that an MSI-positive cell line derived from Burkitt's lymphoma is proficient in MMR.